Towards configurationally stable bisindolylmaleimide cyclophanes: potential tools for investigating protein kinase function.

Article abstract of DOI:10.1039/b400078a

The effect of macrocycle size and substitution on the configurational stability of some bisindolylmaleimide cyclophanes was determined.

Full text of DOI:10.1039/b400078a

Towards configurationally stable bisindolylmaleimide cyclophanes:
potential tools for investigating protein kinase function
Stephen Bartlett and Adam Nelson*
Department of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds,
UK LS2 9JT; Fax: +44 (0)113 343 6565; Tel: +44 (0)113 343 6502. E-mail: adamn@chem.leeds.ac.uk
Received (in Cambridge, UK) 7th January 2004, Accepted 10th March 2004
First published as an Advance Article on the web 2nd April 2004
The effect of macrocycle size and substitution on the configura-
tional stability of some bisindolylmaleimide cyclophanes was
determined.
were 64 : 36 (n = 10), 42 : 58 (n = 9) and > 99 : < 1 (n =
6–8).
Above room temperature, the NMR spectra of 6d (n = 9) and 6e
(n = 10) broadened dramatically. The rate of exchange between the
anti and syn conformers could be determined as a function of
temperature by comparison of experimental and simulated spec-
tra.† The barriers to interconversion between the conformers
showed that macrocycle size had an important effect on the
configurational stability of the cyclophanes 6: at 298 K, the barriers
for isomerisation of the anti confomers were 66.6 kJ mol21 (for 6e,
n = 10) and 70.8 kJ mol21 (for 6d, n = 9) (Table 2).
The 1H NMR spectra of 6a–c (n = 6–8), recorded in d8-toluene
at 373 K, were not broadened, suggesting that racemisation‡ of 6a–
c was rather more difficult. Indeed, analysis of the cyclophanes 6a
(n = 6) and 6c (n = 8) by chiral analytical HPLC at 298 K revealed
two peaks, demonstrating that the half-lives of the enantiomeric
anti conformers were greater than the separation of the peaks (10
Staurosporine, 1, is a potent broad spectrum inhibitor of many
protein kinases.1 However, despite its potency, its lack of
specificity limits its value as a tool for studying kinase function.
Nonetheless, staurosporine has been a useful lead for the discovery
of selective kinase inhibitors. For example, some bisindolylmalei-
mides, in which the planarity of the indolocarbazole ring system has
been broken, are potent and selective inhibitors of particular
kinases:2 the bisindolylmaleimide3 2 selectively inhibits the b
isoforms of protein kinase C (PKCb) and can produce significant
improvements in diabetic retinopathy, nephropathy, neuropathy
and cardiac dysfunction.4
Table 1 Synthesis of the bisindolylmaleimides 5 and 6
Using procedures which were amenable to automation, we
prepared a series of [2.n]metacyclophanes 5 and 6 in which the
length of the tether, n, and the substituent, R, were varied (see Table
1). The effect of R and n on the barrier to interconversion between
the limiting diastereomeric (syn and anti) conformers was investi-
gated (Fig. 1).
Starting
material
Yield
(%)
Entry
n
R
Product
1
2
3
4
5
6
7
8
9
3
3
3
3
3
4
4
4
4
4
6
7
8
9
10
6
7
8
9
10
H
H
H
H
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
69
73
62
74
71
44
76
62
53
63
The 500 MHz 1H NMR spectra of the bisindolylmaleimides 5 (R
= H) revealed that their conformations were in fast exchange on
the NMR timescale at 298 K. However, in the 1H NMR spectrum
of 5a recorded at low temperature, the protons in its six-atom tether
(e.g. NCHAHB) were revealed to be diastereotopic. Molecular
modelling studies using density functional theory (B3LYP/6-31G*)
revealed that the syn conformer of 5a was > 20 kJ mol21 less stable
than that its anti confomer; the diastereotopicity must stem,
therefore, from slow interconversion between the two enantiomeric
anti conformers. The barrier to racemisation of 5a was 36.6 kJ
mol21 in CD2Cl2 at 203 K (Table 2). The NMR spectra of 5b–e
were also recorded at 200 K; however, the diastereotopicity of the
protons in the tether was not revealed, suggesting that inter-
conversion was faster in these cases. In each case, we propose that
the syn conformer is an intermediate in the racemisation of the anti
conformer.
H
Me
Me
Me
Me
Me
10
In contrast, the 500 MHz 1H NMR spectra of the macrocycles 6
were in slow exchange at room temperature. The ratios of the syn
and anti conformers were determined as a function of temperature
by careful analysis of the NMR spectra: the benzylic protons are
enantiotopic in the syn conformer and diastereotopic in the anti
conformer. Shorter tethers selectively destabilised the syn con-
former: for the macrocycles 6, the ratios of anti and syn conformers
Fig. 1 Limiting conformations of bisindolylmaleimides.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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C h e m . C o m m u n . , 2 0 0 4 , 1 1 1 2 – 1 1 1 3

Table 2 Barriers to interconversion between conformers of the cyclophanes 5 and 6
Equilibration
between…
Ratio
at 298 K
n
R
DG°a/kJ mol21
DGf‡a/kJ mol21
DGb‡a/kJ mol21
t1a,b
2
5a
6a
6c
6d
6
6
8
9
H
anti " ent-anti
anti " ent-anti
anti " ent-anti
anti " syn
50 : 50c,d
50 : 50c,g
50 : 50c,g
42 : 58i
0
0
0
36.6e
> 90
> 90
70.8j
36.6e
> 90
< 0.3 msf
> 10 minh
> 10 minh
290 ms
Me
Me
Me
> 90
20.82
71.7j
‡
DH° = 25.1 kJ mol21
DHf‡ = 68.9 kJ mol21
DHb = 74.0 kJ mol21
DS° = 214.1 J mol21 K21 DSf‡ = 26.5 J mol21 K21 DSb = 7.6 J mol21 K21
‡
6e 10 Me
anti " syn
anti " syn
64 : 36i
35 : 65i
1.5
66.6j
65.1j
50 ms
‡
DH° = 24.4 kJ mol21
DHf‡ = 66.7 kJ mol21
DHb = 71.1 kJ mol21
DS° = 220.0 J mol21 K21 DSf‡ = 0.2 J mol21 K21
DSb = 20.2 J mol21 K21
‡
7
—
Me
21.5
51.5j
53.0j
< 0.12 ms
a At 298 K. b Of the anti conformer. c Symmetry demands a 50 : 50 ratio of enantiomeric anti conformers. d Molecular modelling (B3LYP/6-31G*) suggests
that the syn conformer is at least 20 kJ mol21 less stable. e Determined by variable temperature NMR in CD2Cl2. DG‡ is given at the coalesence temperature
(203 K). f On the assumption that DS‡ is small. g Integration of the 500 MHz 1H NMR spectrum revealed a > 99 : < 1 mixture of anti and syn conformers.
h The enantiomeric anti conformers were resolved by chiral analytical HPLC. i Ratio of the anti and syn conformers determined by integration of the 500
MHz 1H NMR spectrum. j Determined by comparison of simulated spectra, generated using gNMR,5 with experimental spectra recorded at several
temperatures in d8-toluene.
min),6 and, hence, that the barrier to racemisation was at least 90 kJ
mol21
tether. The half lifes, t , of the anti conformers of the [2.n]
1
2
.
metacyclophanes 6 increase from 50 ms (with n = 10) to 290 ms
(with n = 9) to greater than 10 min (with n 5 8).
We thank EPSRC for funding, Julie Fisher, Steve Homans and
Stuart Warriner for helpful discussions, Simon Barrett for vt-NMR
experiments, Jacqueline Colley for HPLC analyses and Andrew
Leach for molecular modelling.
The tether had a smaller effect on the configurational stability of
the cyclophanes 6 than did the indolyl 2-methyl groups. The barrier
to isomerisation of the anti conformer of the bisindolylmaleimide 7,
in which the tether had been removed, was 51.5 kJ mol21; in
contrast, the barrier to racemisation of 5a, in which R = H, was just
36.6 kJ mol21
.
Notes and references
† gNMR was used to produce simulated spectra based on populations of the
syn and anti conformers extrapolated from the slow exchange regime.
‡ The cyclophanes 6a–c exclusively ( > 95 : 5) populated the chiral (anti)
conformer.
1 For a review see: U. T. Rüegg and G. M. Burgess, Trends Pharm. Sci.,
1989, 10, 218.
There is a substituent in each of the ortho positions flanking each
[3,3A]bipyrrolyl bond of 7 (the bipyrrolyl unit is shown in black);
given that tetrasubstituted biphenyls 8 (A ≠ B ≠ H; C ≠ D ≠ H)
are generally resolvable,7 it is, perhaps, surprising that the
conformers of bisindolylmaleimides such as 7 are not atropisomers.
However, the internal bond angles of a biphenyl8 (119°) are
markedly wider than those of a bisindolylmaleimide9 (107° and
108° in the crystal structure of 3), so the carbons which are ortho to
the bipyrrolyl bond are further apart (2.96 and 3.30 Å8) than for
biphenyl7 (2.92 Å). In addition, small size of the maleimide oxo
group can be rivalled by only a hydrogen atom (compare the length
of its carbonyl bond, 1.21 Å, with bonds to other “small”
substituents:7 1.39 Å for Ph–F, 1.45 Å for Ph–OH). Furthermore,
conjugation between a maleimide and an indole in the transition
state is far more stabilising than conjugation between two phenyl
rings.
In summary, the addition of indolyl 2-methyl substituents to
bisindolylmaleimides such as 3 is not sufficient for configurational
stability. The effect of indolyl 2-methyls may be exaggerated by the
presence of a tether between the nitrogen atoms of the indoles
(?6). Like other metacyclophanes,10 the activation energy for
interconversion between limiting conformers (anti and syn con-
fomers in this case) is critically dependent on the length of the
2 R. A. Bit, P. D. Davis, L. H. Elliott, W. Harris, C. H. Hill, E. Keech, G.
Kumar, A. Maw, J. S. Nixon, D. R. Vessey, J. Wadsworth and S. E.
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Boissin, E. Boursier, F. Loriolle, L. Duhamel, D. Charon and J.
Kirilovsky, J. Biol. Chem., 1991, 266, 15771.
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McDonald III, D. A. Neel, C. J. Rito, U. Singh, L. E. Stramm, A.
Melikian-Badalian, M. Baevsky, L. M. Ballas, S. E. Hall, L. L.
Winneroski and M. M. Faul, J. Med. Chem., 1996, 39, 2664; W. F.
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4 K. J. Way, N. Katai and G. L. King, Diabetic Med., 2001, 18, 945.
5 C. P. Budzelaar, gNMR, ver. 5.0, University of Nijmegen, The
Netherlands.
6 J. Veciana and M. I. Crespo, Angew. Chem., Int. Ed. Engl., 1991, 30,
74.
7 R. Adams and H. C. Yuan, Chem. Rev., 1933, 12, 261; M. Charton, J.
Org. Chem., 1977, 42, 2528.
8 J. Trotter, Acta Crystallogr., 1981, 14, 1135.
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Hurst, E. Keech and S. E. Turner, J. Med. Chem., 1992, 35, 177.
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C h e m . C o m m u n . , 2 0 0 4 , 1 1 1 2 – 1 1 1 3
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